Emerging evidence this decade suggests that estrogen deprivation may be an important factor in the occurrence of age-related neurodegenerative diseases, such as Alzheimer's disease (AD). This concept is supported by findings that postmenopausalestrogen-replacement therapy (ERT) is associated with a reduction in the incidence of AD. Similarly, women undergoing ERT are reported to have lower mortality from stroke. These studies have identified a neuroprotective role of estrogens in humans, which has now been confirmed in animal's models. This laboratory has demonstrated the potent neuroprotective effects of estradiol in both animal and cell culture models following exposure to neurotoxic insults. To date, the molecular mechanisms ofneuroprotective effects of estrogens remain unclear. Studies have shown, however, that the neuroprotective effects are independent of the estrogenic potency of the molecule. This and other findings have provided evidence that the protection is not mediated through classical estrogen receptors, but is likely non-genomic, possibly mediated through signal transduction pathways. Based on estrogen's known effects on protein kinase C-mediated signaling, and reports of PKC-mediated neuroprotection; we have initiated studies to examine the role of PKC in estrogen-induced neuroprotection. Preliminary data presented herein demonstrate that neuroprotection by estradiol, in an immortalized hippocampal cell line exposed to glutamate, is mimicked and enhanced by PKC down-regulation or by exposure to PKC inhibitors, and that that PKC down-regulation/inhibition is in itself neuroprotective. We also demonstrate that estradiol exposure rapidly inhibits the specific activity, and alters the intracellular distribution, of at least one PKC isozyme in a dose-dependent manner that correlates with estradiol's neuroprotective effects. Additional preliminary data also implicate the MAPK signaling pathway and the nuclear factor-kB as possible downstream mediators of estradiol's effects on PKC. Our findings have led us to hypothesize that PKC down-regulation mediates a neuroprotective signal from estrogen, which is then propagated though the MAPK pathway, and ultimately results in altered gene expression beneficial to cell survival. The studies proposed in this application are designed to critically evaluate the role of PKC in mediating the neuroprotective effects of estrogen. The specific aims of these studies are as follows: 1) We will characterize the role of PKC in estrogen-mediated neuroprotection by examining the effect of estrogen on PKC activity and distribution, and by examining the effect of PKC modulation on neuroprotection. We will also assess the role of the MAPK signaling pathway, and NFkB, which appear to play important roles in the PKC-mediated enhancement of estrogen neuroprotection. 2) We will examine the generalizability of the PKC-mediated enhancement of estrogen neuroprotection to additional neurotoxic insults, and to extend these findings to a more physiologically relevant primary neuronal cell model. 3) Finally, using in vivo regulators of PKC isozymeactivity, we will test the hypothesis that inactivation of PKC enhances the neuroprotective effects of estrogen in a rat model of ischemia. These studies have implications for designing neuroprotective agents to combat neurodegenerative diseases and neuronal insults.